Sustained release formulations

ABSTRACT

The present invention relates broadly to the field of sustained release formulations. More specifically, the invention describes compositions and methods relating to formulating proteins and/or peptides with purified gallic acid esters. In one example, the gallic acid ester is PentaGalloylGlucose (PGG) and in anther example the gallic acid ester is epigallocatechin gallate (EGCG).

This Application claims the benefit of priority to U.S. ProvisionalApplication Ser. No. 60/565,247, filed Apr. 23, 2004.

FIELD OF THE INVENTION

The present invention relates broadly to the field of sustained releaseformulations. More specifically, the invention describes compositionsand methods relating to formulating proteins and/or peptides withpurified gallic acid esters. In one example, the gallic acid ester isPentaGalloylGlucose (PGG), where gallic acid is also known as 3,4,5trihydroxybenzoic acid and in another example the gallic acid ester isepigallocatechin gallate (EGCG).

BACKGROUND

To achieve continuous delivery of the protein or peptide in vivo, asustained release or sustained delivery formulation is desirable toavoid the need for repeated administrations. One approach for sustaineddrug delivery is by microencapsulation, in which the active ingredientis enclosed within a polymeric membrane to produce microparticles.

It has been shown that one can encapsulate a biologically active orpharmaceutically active agent within a biocompatible, biodegradable wallforming material such as a polymer, to provide sustained or delayedrelease. In these methods the agent or drug is typically dissolved,dispersed or emulsified, using stirrers, agitators, or other dynamicmixing techniques, in one or more solvents containing the wall formingmaterial. The solvent is then removed resulting in the formation ofmicroparticles encapsulating the agent or drug. These microparticles canthen be administered to a patient.

The importance of biocompatible and/or biodegradable polymers ascarriers for parenteral drug delivery systems is now well established.Biocompatible, biodegradable, and relatively inert substances such aspoly(lactide) (PLA) or poly(lactide-co-glycolide) (PLGA) microspheres orfilms containing the active agent to be administered are commonlyutilized sustained-release devices (for review, see M. Chasin,Biodegradable polymers for controlled drug delivery. In: J. O. HollingerEditor, Biomedical Applications of Synthetic Biodegradable Polymers CRC,Boca Raton, Fla. (1995), pp. 1-15; T. Hayashi, Biodegradable polymersfor biomedical uses. Prog. Polym. Sci. 19 4 (1994), pp. 663-700; andHarjit Tamber, PaIl Johansen, Hans 10 P. Merkle and Bruno Gander,Formulation aspects of biodegradable polymeric microspheres for antigendelivery Advanced Drug Delivery Reviews, Volume 57, Issue 3, 10 Jan.2005, Pages 357-376).

However, there still exist many challenges to the design of deliverysystems for active agents. A basic requirement for such delivery systemsis that the materials used are acceptable for parenteral application. Asmentioned above, it is desirable that the materials used arebiodegradable for formulations intended for repeated administration.Another generally desirable quality is biocompatibility: the materialsshould be tolerated well and biodegradation should produce innocuouscompounds that are either eliminated from the body or incorporated inthe intermediary metabolism. The list of materials used generally formanufacture of parenteral preparations is limited and is shorter stillfor parenteral protein formulations.

Another desirable attribute is sufficiently good control of the releaseof the encapsulated active agent. It is generally important to maintainthe concentration of the active agent within an effective window for atime period sufficient to achieve the desired effect and to avoidexcessive concentrations, which may lead to side effects or untowardresults. It is often difficult to achieve the desired release kineticswith monolithic microparticles as the fraction of the active agentreleased within the first day after administration is often dependent onthe loading level of the drug.

Yet another desirable characteristic of sustained delivery technologies,particularly when applied to the delivery of macromolecules, is that theintegrity of the active agent is maintained during manufacture. This isoften a difficult challenge as most protein and peptide drugs aredependent on a three dimensional conformation for their bioactivity andthat conformation can easily be compromised. For example, most of thepolymers that are used to manufacture controlled release parenteralpreparations are not soluble in water and consequently the protein orpeptide is exposed to an organic solvent in the encapsulation step.Examples of other undesireable stresses that are associated withmanufacturing of controlled release preparations that may compromise theintegrity of any particular active agent are high shear forces used toform droplets of the polymer solution in an continuous phase, exposureto polymerization reactions, high temperatures and undesirably low orhigh pH values.

Another desirable attribute of sustained release modalities is that theintegrity of the active agent, particularly proteins or peptides, isretained within the microparticles during release. Depending on thechosen duration of release, this period can be from a few days up toseveral months. For conventional polymer matrix systems formed of PLGAthe acidic microenvironment formed during biodegradation of the polymermay degrade active agents incorporated therein during in vitro and invivo incubation.

The prior art describes various sustained delivery compositions andmethods for making them, for example, U.S. Pat. Nos. 5,916,597;5,019,400; 5,922,253; and 6,531,154. The in vivo release of incorporatedactive agents from biocompatible and biodegradable polymers is, in manycases, initially high or low, and therefore non-uniform throughout thelife of the delivery device. Additionally, microencapsulation withpolymers tends to provide long term sustained delivery of peptidesranging from two weeks to nine months or longer whereas there is a needfor shorter term delivery profiles for certain medicaments. Thus, thereis a need in the art for sustained release compositions with releaseprofiles of less than about a week or two.

SUMMARY OF THE INVENTION

The present invention provides pharmaceutical compositions comprising astable sustained release complex composed of a protein and/or peptideand a gallic acid ester that allow for sustained delivery of the proteinor peptide in vivo upon administration of the complex. Accordingly, thecomplex of the invention can permit continuous delivery of apharmaceutically active peptide to a subject for periods of time lessthan about one or two weeks.

The complex of the invention is formed by combining a protein or peptideand a gallic acid ester under conditions such that a complex is formed.In a preferred embodiment, the complex is a salt of the gallic acidester and protein or peptide. The complex is typically poorly soluble inwater and can be purified from various aqueous solutions. As the complexis in the form of a solid (e.g., a paste, granules, a powder or alyophilizate), the complex can be prepared for administration to asubject as a stable liquid suspension or semi-solid dispersion.

In one embodiment of the invention, the group suitable for use informing a complex with a peptide or protein is a gallic acid ester.Preferably, the ester itself is formed by a linkage of the acid group ofgallic acid to an alcohol moiety on another compound such as a sugar. Ina particular embodiment, the gallic acid ester is PentaGalloylGlucose(PGG), where the gallic acid is also known as 3,4,5-trihydroxybenzoicacid. In another embodiment, the gallic acid ester is EpigallocatechinGallate (EGCG).

DETAILED DESCRIPTION

The present invention relates to compositions comprising a sustainedrelease complex composed of a protein or peptide and a gallic acidester, methods of making such compositions and methods of using suchcompositions. While gallic acid esters are a known component of tannicacid, the use of a highly purified component of tannic acids such asparticular gallic acid esters to make a salt with peptides andpolypeptides to create a sustained release formulation as describedherein, has not been described. The advantages of the compositions ofthe invention include the delivery of the peptide or protein portion ofthe complex, either systemically or locally, for a controlled periods(e.g., typically less than about one or two weeks). Delivery for longerperiods of time is also contemplated.

As used herein, the terms “protein” and “peptide” are understood toinclude polymers of amino acids linked by amide bonds. Typically, apeptide will be composed of less than about 50 amino acids, moretypically less than about 30 amino acid residues and even moretypically, less than about 20 amino acid residues. Whereas a proteinwill typically be composed of more than 50 amino acids and will havestructure and biological activity. The protein's biological activity canbe enzymatic or it may be a binding activity that confers conformationchanges. These terms are further intended to encompass analogues andderivatives that mimic the chemical structure of the components of theprotein or peptides. Examples of analogues include peptides or proteinscontaining one or more non-natural amino acids. Examples derivativesinclude peptides or proteins containing amino acid side chain(s),peptide backbone, and/or amino- or carboxy-terminus that have beenderivatized.

Peptides suitable for formulation according to the invention include butare not limited to enfuvirtide (sold by Trimeris and Roche as Fuzeon®),Angiotensin, Amylin, ACTH, renin substrate, Cecropin A-Melittin amide,Cecropin B, Magainin 1, Renin Inhibitor Peptide, Bombesin, Osteocalcin,Bradykinin, B 1 Inhibitor Peptide, Bradykinin peptide antagonists,including bradykinin peptide antagonists disclosed in U.S. patentapplication Ser. No. 10/972,236, filed on Oct. 21, 2004,Kallidin,Calcitonin, Cholecystokinin, Corticotropin Releasing Factor, DynorphinA, Endomorphin, Sarafotoxin, Enkephalin, Exendin, Fibrinopeptide,Galanin, Gastrin, Gastrin Releasing Peptide, Glucagon-Like Peptide,Growth Hormone Releasing Factor, OVA Peptide, LuteinizingHormone-Releasing Hormone, Atrial Natriuretic Peptide, MelaninConcentrating Hormone, Brain Natriuretic Peptide, Vasonatrin,Neurokinin, Neuromedin, Neuropeptide Y, Neurotensin, Orexin, Oxytocin,Vasopressin, Parathyroid Hormone Peptide, Prolactin Releasing Peptide,Somatostatin, Somatostatin Tumor Inhibiting Analog, ThyrotropinReleasing Hormone, and variants and derivatives thereof (see also,Latham, (1999) Nat. Biotech., 17:755).

Proteins that can be formulated according to the invention include butare not limited to Flt3 ligand, CD40 ligand, erythropoietin,thrombopoeitin, calcitonin, Fas ligand, ligand for receptor activator ofNF-kappa B (RANKL), TNF-related apoptosis-inducing ligand (TRAIL),ORK/Tek, thymic stroma-derived lymphopoietin, granulocyte colonystimulating factor, granulocyte-macrophage colony stimulating factor,mast cell growth factor, stem cell growth factor, epidermal growthfactor, RANTES, growth hormone, insulin, insulinotropin, insulin-likegrowth factors, parathyroid hormone, nerve growth factors, glucagon,interleukins 1 through 18, colony stimulating factors, lymphotoxin-B,tumor necrosis factor, leukemia inhibitory factor, oncostatin-M, andvarious ligands for cell surface molecules Elk and Hek (such as theligands for eph-related kinases, or LERKS). Descriptions of making suchproteins proteins may be found in, for example, Human Cytokines:Handbook for Basic and Clinical Research, Vol. II (Aggarwal andGutterman, Eds. Blackwell Sciences, Cambridge Mass., 1998); GrowthFactors: A Practical Approach (McKay and Leigh, Eds. Oxford UniversityPress Inc., New York, 1993) and The Cytokine Handbook (A W Thompson,ed.; Academic Press, San Diego Calif.; 1991).

Receptors for any of the aforementioned proteins can also be formulatedaccording to the invention, provided that they are soluble portions ofthe molecule suitable for administration to a subject. Examples includethe receptors for both forms of tumor necrosis factor receptor (referredto as p55 and p75), Interleukin-1 receptors (type 1 and 2),Interleukin-4 receptor, Interleukin-15 receptor, Interleukin-17receptor, Interleukin-18 receptor, granulocyte-macrophage colonystimulating factor receptor, granulocyte colony stimulating factorreceptor, receptors for oncostatin-M and leukemia inhibitory factor,receptor activator of NF-kappa B (RANK), receptors for TRAIL, andreceptors that comprise death domains, such as Fas or Apoptosis-InducingReceptor (AIR). A particularly preferred receptor is a soluble form ofthe IL-I receptor type II; such proteins are described in U.S. Pat. No.5,767,064, incorporated herein by reference in its entirety.

Other proteins that can be formulated according to the invention includesoluble variants of cluster of differentiation antigens (referred to asCD proteins), for example, those disclosed in Leukocyte Typing VI(Proceedings of the VIth International Workshop and Conference;Kishimoto, Kikutani et al., Eds. Kobe, Japan, 1996), or CD moleculesdisclosed in subsequent workshops. Examples of such molecules includeCD27, CD30, CD39, CD40; and ligands thereto (CD27 ligand, CD30 ligandand CD40 ligand). Several of these are members of the TNF receptorfamily, which also includes 41BB and OX40; the ligands are often membersof the TNF family (as are 41BB ligand and OX40 ligand); accordingly,members of the TNF and TNFR families can also be produced using thepresent invention.

Enzymatically active proteins can also be formulated according to theinvention. Examples include metalloproteinase-disintegrin familymembers, various kinases, glucocerebrosidase, alpha-galactosidase A,superoxide dismutase, tissue plasminogen activator, Factor VIR, FactorIX, apolipoprotein E, apolipoprotein A-I, globins, an IL-2 antagonist,alpha-1 antitrypsin, TNF-alpha Converting Enzyme, and numerous otherenzymes. Ligands for enzymatically active proteins can also beformulated by applying the instant invention.

The inventive compositions and methods are also useful for formulationof other types of proteins, including immunoglobulin molecules orportions thereof, and chimeric antibodies (i.e., an antibody having ahuman constant region couples to a murine antigen binding region) orfragments thereof. Numerous techniques are known by which DNA encodingimmunoglobulin molecules can be manipulated to yield DNAs capable ofencoding recombinant proteins such as single chain antibodies,antibodies with enhanced affinity, or other antibody-based proteins(see, for example, Larrick et al., 1989, Biotechnology 7:934-938;Reichmann et al., 1988, Nature 332:323-327; Roberts et al., 1987, Nature328:731-734; Verhoeyen et al., 1988, Science 239:1534-1536; Chaudhary etal., 1989, Nature 339:394-397). The term humanized antibody alsoencompasses single chain antibodies. See, e.g., Cabilly et al., U.S.Pat. No. 4,816,567; Cabilly et al., European Patent No. 0,125,023 B1;Boss et al., U.S. Pat. No. 4,816,397; Boss et al., European Patent No.0,120,694 B1; Neuberger, M. S. et al., WO 86/01533; Neuberger, M. S. etal., European Patent No. 0,194,276 B1; Winter, U.S. Pat. No. 5,225,539;Winter, European Patent No. 0,239,400 B1; Queen et al., European PatentNo. 0 451 216 B1; and Padlan, E. A. et al., EP 0 519 596 A1. Forexample, the invention can be used to formulate human and/or humanizedantibodies that immunospecifically recognize specific cellular targets,e.g., any of the aforementioned proteins, the human EGF receptor, theher-2/neu antigen, the CEA antigen, Prostate Specific Membrane Antigen(PSMA), CD5, CD11a, CD18, NGF, CD20, CD45, Ep-cam, other cancer cellsurface molecules, TNF-alpha, TGF-betal, VEGF, other cytokines, alpha 4beta 7 integrin, IgEs, viral proteins (for example, cytomegalovirus),etc., to name just a few.

Various fusion proteins can also be formulated according to theinvention. A fusion protein is a protein, or domain or a protein (e.g. asoluble extracellular domain) fused to a heterologous protein orpeptide. Examples of such fusion proteins include proteins expressed asa fusion with a portion of an immunoglobulin molecule, proteinsexpressed as fusion proteins with a zipper moiety, and novelpolyfunctional proteins such as a fusion proteins of a cytokine and agrowth factor (i.e., GM-CSF and IL-3, MGF and IL-3). WO 93/08207 and WO96/40918 describe the preparation of various soluble oligomeric forms ofa molecule referred to as CD40L, including an immunoglobulin fusionprotein and a zipper fusion protein, respectively; the techniquesdiscussed therein are applicable to other proteins. Another fusionprotein is a recombinant TNFR:Fc, also known as “etanercept.” Etanerceptis a dimer of two molecules of the extracellular portion of the p75 TNFalpha receptor, each molecule consisting of a 235 amino acidTNFR-derived protein that is fused to a 232 amino acid Fc portion ofhuman IgG1. In fact, any of the previously described molecules can beexpressed as a fusion protein including but not limited to theextracellular domain of a cellular receptor molecule, an enzyme, ahormone, a cytokine, a portion of an immunoglobulin molecule, a zipperdomain, and an epitope.

As used herein, the term “gallic acid ester” is intended to refer to amolecule that can complex with a protein or peptide to form a sustainedrelease complex. In one example, the gallic acid ester molecule is aPentaGalloylGlucose (PGG, also referred to in the art as 5GG). It isunderstood that the PGG molecule can have one galloyl group, two galloylgroups, three galloyl groups or four galloyl groups. In addition, it isunderstood that glucose can be replaced with another carbon backbone,such as an alcohol or polyol, e.g., glycerol, ethylene glycol or anysugar group suitable for use. In another example, EpigallocatechinGallate (EGCG) is the gallic acid ester molecule useful in the inventionto make a salt with a peptide or protein. EGCG is an anti-oxidantpolyphenol flavonoid isolated from green tea. The EGCG ester is attachedto a ring structure that is not a sugar, in contrast to PGG. Further, itis understood that the gallic acid ester can assume differentstereochemical forms. For example, PGG can be in either alpha or betaforms. One of skill in the art will be able, for the teachings herein,to identify appropriate gallic acid ester molecules for use in thecompositions and methods of the invention.

As used herein, the term “sustained release complex” is intended torefer to a physically and chemically stable complex that forms uponappropriate combining of a protein or peptide and gallic acid esterdescribed herein. This complex typically takes the form of a precipitatethat is produced upon combining aqueous or non-aqueous preparations ofthe protein or peptide and gallic acid ester.

As used herein, the term “sustained delivery” is intended to refer tocontinual delivery of a pharmaceutical agent in vivo over a period oftime following administration. Sustained delivery of the agent can bedemonstrated by, for example, the continued therapeutic effect of theagent over time. Alternatively, sustained delivery of the agent may bedemonstrated by detecting the presence of the agent in vivo over time.In one embodiment, the sustained delivery is less than a week and can beless than four days. However, it is also contemplated that the sustaineddelivery can be for periods longer than one week using the compositionsof the invention, including more than two weeks.

The formation of a PGG or EGCG complex with a peptide or protein atdifferent pH's can affect the period of drug delivery. As shown below inExamples 4 and 5, formation of a PGG complex with a peptide at pH 7.0results in longer duration in serum of the complex, i.e., about a week,than those complexes formed at pH 7.6 and pH 8.6, i.e., less than aweek. Thus, it is an embodiment of the invention that the duration ofdrug delivery is controlled in part by the pH at which the complex isformed. A representative pH range is 6.0 to 9.0, and the ranges of pH6.5 to 8.6, pH 7.0 to 8.6 are also suitable. One of skill in the artwould readily understand that other pH's may be suitable and given theteachings of the invention, it is no more than routine experimentationto determine what pH best suits the desired drug delivery profile of aparticular drug complexed with the tannic acid esters of the invention.

One aspect of the present invention pertains to a pharmaceuticalcomposition comprising a sustained release complex of a pharmaceuticallyactive agent such as a protein or peptide and a gallic acid ester. Thepharmaceutical compositions of the invention permit sustained deliveryof a protein or peptide to a subject in vivo after administering thecomposition to the subject, wherein the duration of the sustaineddelivery can be varied depending upon the solubility of peptide andgallic acid ester complex. For example, in one embodiment, the sustainedrelease complex provides sustained delivery of a pharmaceutically activeagent to a subject for at less than one week after a pharmaceuticalcomposition of the invention is administered to the subject. In anotherembodiment, the sustained release complex provides sustained delivery ofa protein or peptide to a subject for less than four days. Formulationsthat provide sustained delivery for longer or shorter durations are alsoencompassed by the invention, such as formulations that providecontinuous delivery for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days ora week and the like. Likewise, it is contemplated that thesecompositions can be formulated such that they provide continuous drugdelivery for over one week, and up to two weeks, or more.

Any size amino acid chain may be suitable for use in the complex as longas the protein or peptide has the ability to form a sustained releasenoncovalent complex with the gallic acid ester upon combination of thetwo.

In addition to the sustained release complex, the pharmaceuticalformulations of the invention can comprise additional pharmaceuticallyacceptable carriers and/or excipients. As used herein, “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like that are physiologically compatible.Preferably, the carrier is suitable for intravenous, intramuscular,subcutaneous or parenteral administration (e.g., by injection).Excipients include pharmaceutically acceptable stabilizers anddisintegrants.

In addition to pharmaceutical formulations of peptides complexed with agallic acid ester, the invention further encompasses packagedformulations containing such complexes and syringes containing suchcomplexes. In another embodiment, the invention provides a syringehaving a lumen, wherein a sustained release complex of a protein orpeptide and a gallic acid ester is included in the lumen.

The complex of the invention is prepared by combining the protein orpeptide and the gallic acid ester under conditions such that a sustainedrelease complex of the protein or peptide and the gallic acid esterforms. Accordingly, another aspect of the invention pertains to methodsfor preparing pharmaceutical formulations. In one embodiment, the methodcomprises providing a protein or peptide and a gallic acid ester,combining the protein or peptide and the gallic acid ester underconditions such that a complex of the protein or peptide and the gallicacid ester forms, and preparing a pharmaceutical formulation comprisingthe complex.

For example, a solution of the protein or peptide and a solution of thegallic acid ester are combined until a sustained release complex of theprotein or peptide and the gallic acid ester precipitates out ofsolution. In certain embodiments, the solutions of the protein orpeptide and the gallic acid ester are aqueous solutions. The amounts ofprotein or peptide and gallic acid ester necessary to achieve thesustained release complex may vary depending upon the particular proteinor peptide and gallic acid ester used, the particular solvent(s) usedand/or the procedure used to achieve the complex. Often, the protein orpeptide also will be in excess on a weight/weight basis, as demonstratedin the Examples.

Once the protein or peptide/gallic acid ester complex precipitates outof solution, the precipitate can be removed from the solution by meansknown in the art, such as filtration, centrifugation and the like. Therecovered material then can be dried and the solid can be milled orpulverized to a powder by means known in the art. Alternatively, thepaste can be frozen and lyophilized to dryness. The powder form of thecomplex can be dispersed in a carrier solution to form a liquidsuspension or semi-solid dispersion suitable for injection. Accordingly,in various embodiments, a pharmaceutical formulation of the invention isa lyophilized solid, a liquid suspension or a semi-solid dispersion.

In another embodiment, the pharmaceutical formulation of the inventionis sterile formulation. For example, following formation of thesustained release complex, the complex can be sterilized by gammairradiation or electron beam sterilization. Pharmaceutical formulations,including powders, liquid suspensions, semi-solid dispersions,lyophilized solids, and sterilized forms thereof (e.g., by gammairradiation or sterile filtration), prepared according to the methods ofthe invention, are also encompassed by the invention.

As used herein, the term “subject” is intended to include is intended toinclude warm-blooded animals, preferably mammals, most preferablyhumans.

As used herein, the term “administering to a subject” is intended torefer to dispensing, delivering or applying a composition (e.g.,pharmaceutical formulation) to a subject by any suitable route fordelivery of the composition to the desired location in the subject,including delivery by either the parenteral or oral route, intramuscularinjection, subcutaneous/intradermal injection, intravenous injection,buccal administration, transdermal delivery and administration by therectal, colonic, vaginal, intranasal or respiratory tract route.

The following examples are merely representative embodiments and notmeant to be limiting as to the full scope of the invention.

EXAMPLE 1

Example 1 provides a description of a preparation of Peptide B-PGG salt(1:1 molar ratio of Peptide B (DOrn Lys Arg Pro Hyp Gly Cpg Ser DticCpg) to PGG). A stock solution of PGG was made by dissolving 94 mg ofPGG in 2 ml of NaOH solution (concentration of NaOH from 0.10 to 0.20 N)following by filtering it through a 0.2 um filter. To a stock solutionof PGG (1.56 mL) was added sequentially a solution of 109.4 mg ofPeptide B acetate salt in 0.8 mL water with stirring and a precipitateformed. The precipitate was recovered by centrifugation.

The supernatant was decanted and the precipitate was washed with 0.5 mLwater 3 times. The precipitate was dried in vacuum at approximately30-35° C. for approximately 20 hours to yield 125 mg (76%). The PeptideB-PGG salt was an off-white powder.

EXAMPLE 2

Salts of Peptide A-PGG and tannate were made in a similar way to PeptideB-PGG in Example 1. Peptide A was Acetyl Lys Lys Arg Pro Hyp Gly Cpg SerDtic Cpg.

EXAMPLE 3

The tables 1 and 2 below list peptide content and solubility of PeptideA (Acetyl Lys Lys Arg Pro Hyp Gly Cpg Ser Dtic Cpg) and Peptide B (DOrnLys Arg Pro Hyp Gly Cpg Ser Dtic Cpg)salts with tannate and PGG in waterand PBS. The data showed that the PGG peptide salt has higher peptidecontent than the tannate.

The PGG precipitates have higher PBS solubility than the tannate.

EXAMPLE 4

The study of the effect of salt formation pH (i.e. concentration levelof NaOH) on the yield, peptide content and solubility of Peptide B-PGGsalt was investigated. Four Peptide B-PGG salts at pH 7.0, 7.2, 7.6 and8.6 were prepared and isolated. Their solubility in water and PBS, andalso their peptide content were then determined. These resultsdemonstrate (Table 3) that aqueous solubility, yield of salt formationand peptide content increase with increasing pH during salt formation.

EXAMPLE 5

This example describes sustained release of Peptide B/PGG and PeptideB/tannate salts in rats. The rat pharmacokinetics (PK) studies wereperformed by a single subcutaneous injection (10 mg/kg dose) of PeptideB/PGG salts and Peptide B/tannate salt suspended in TRIS buffer; and aPBS solution of Peptide B acetate as a control group. The PK resultsshowed one-week sustained release for Peptide B/tannate salt and PeptideB-PGG salt that prepared at pH 7.0. However, Peptide B-PGG saltsprepared at pH 7.6 and 8.6 showed shorter release duration (around 2-3days) compared to salt prepared at pH 7.0 (up to two weeks).

EXAMPLE 6

A pure anomer (beta-PGG) and a mixture of anomers (alpha+beta-PGG) ofPGG salts of Peptide B (DOrn Lys Arg Pro Hyp Gly Cpg Ser Dtic Cpg) wereprepared by a similar method to that described in Example 1. There wasno significant difference in the aqueous solubility of these salts.Based on aqueous solubility, it is expected that the in vivo sustainedrelease duration for these salts would be similar.

EXAMPLE 7

The following example describes the use of EGCG to make a salt with apeptide, which was tested in an animal pharmacokinetic (PK) study forsustained release. A stock solution of EGCG (Sigma-Aldrich) was made bydissolving 184 mg of EGCG in 2 ml of 0.2 N NaOH followed by filtering itthrough a 0.2 um filter. To a stock solution of EGCG (1.4 mL) was slowlyadded a solution of 138 mg of acetate salt of Peptide B (DOrn Lys ArgPro Hyp Gly Cpg Ser Dtic Cpg) in 1.2 mL water with stirring. Theresulting suspension was stirred for approximately 10-15 minutes at roomtemperature. After centrifugation, the supernatant was decanted and theprecipitate was washed with 1 InL water (3 times by centrifugation anddecantation of supernatant). The precipitate was dried under vacuum atapproximately 30-35° C. for approximately 20 hours to yield 218 mg (88%)of Peptide B-EGCG salt as an off-white powder.

The peptide content of the Peptide B/EGCG salts were 47-50%. The aqueoussolubility for the salt with 1:3 molar ratio of peptide to EGCG is <0.5mg/ml in water and <0.05 mg/ml in PBS, and for 1:2 molar ratio ofpeptide to EGCG, solubility is approximately 1 mg/ml in water andapproximately 0.3 mg/ml in PBS. A rat PK study was performed using asingle sc injection (10 mg/kg dose) of '593/EGCG salt suspended in TRISbuffer, pH7.0. The PK result showed sustained release of Peptide B formultiple days with the blood level >26 ng/ml at 24 hours, then adecrease to approximately 5 ng/ml at 96 hours. TABLE 1 Peptide APrecipitates Peptide Peptide Conjugated Peptide solubility Yield puritycontent salt purity (mg/mL) at RT Salt name (%) (%) (%) (%) H₂O PBS PGG˜78 >98 50 >97% ˜0.03 ˜0.2 (α + β) Tannate ˜75 >98 32 N/A ˜0.01 ˜0.05

TABLE 2 Peptide B Precipitates Peptide Peptide Conjugated Peptidesolubility Yield purity content salt purity (mg/mL) at RT Salt name (%)(%) (%) (%) H2O PBS PGG ˜75 >98 44 >97% ˜0.2 ˜0.08 (α + β) Tannate˜75 >98 28 N/A ˜0.2 ˜0.01

TABLE 3 Peptide B precipitates formed at different pH Pep- Saltformation tide condition con- Peptide B Conc. of Conc. of Yield tent(mg/ml) Salts PGG NaOH pH (%) (%) In H₂O In PBS Peptide 0.05 M 0.10 N7.0 Approx. 46.5 Approx. Approx. B-PGG 70 0.2 0.08 Peptide 0.05 M 0.12 N7.2 Approx. 50.0 Approx. Approx. B-PGG 75 0.1 0.15 Peptide 0.05 M 0.15 N7.6 Approx. 53.0 <0.1 Approx. B-PGG 92 0.2 Peptide 0.05 M 0.20 N 8.6Approx. 56.5 Approx. B-PGG 96 0.4

While the present invention has been described in terms of preferredembodiments, it is understood that variations and modifications willoccur to those skilled in the art. Therefore, it is intended that theappended claims cover all such equivalent variations which come withinthe scope of the claims.

Each of the patents, applications and articles cited hereinabove(hereinafter, “references”), and each document cited or referencedtherein, including during the prosecution of any of the patents and/orapplications cited herein (“patent cited documents”), and anymanufacturer's instructions or catalogues for any products cited hereinor mentioned in any of the references and in any of the patent citeddocuments, are hereby incorporated herein by reference. Furthermore, alldocuments cited in this text, and all documents cited or referenced indocuments cited in this text, and any manufacturer's instructions orcatalogues for any products cited or mentioned in this text or in anydocument hereby incorporated into this text, are hereby incorporatedherein by reference. Documents incorporated by reference into this textor any teachings therein may be used in the practice of this invention.Documents incorporated by reference into this text are not admitted tobe prior art.

1. A sustained release composition comprising a purified complex of aprotein or peptide and a purified gallic acid ester.
 2. The compositionof claim 1, wherein the complex is a salt of the peptide and the gallicacid ester.
 3. The composition of claim 1, wherein the gallic acid esteris selected from the group consisting of PentaGalloylGlucose (PGG) andepigallocatechin gallate (EGCG).
 4. The composition of claim 3 whereinthe purified gallic acid ester is PentaGalloylGlucose (PGG).
 5. Thecomposition of claim 4, wherein the peptide and PGG salt has a releaseduration in an animal of one week.
 6. The composition of claim 4,wherein the peptide and PGG salt has a release duration in an animal of3 days.
 7. The composition of claim 3 wherein the purified gallic acidester is epigallocatechin gallate (EGCG).
 8. The composition of any ofclaims 2, 3, or 4 wherein said peptide is a B1 peptide antagonist. 9.The composition of claim 8 wherein said peptide is selected from i) DOrnLys Arg Pro Hyp Gly Cpg Ser Dtic Cpg; and ii) Acetyl Lys Lys Arg Pro HypGly Cpg Ser Dtic Cpg wherein DOrn is the D isomer of ornithine, Hyp isTrans-4-hydroxy-proline, Dtic is the D isomer of1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, and Cpg iscyclopentylglycine.
 10. The composition of claim 9 wherein said peptideis selected from i) DOrn Lys Arg Pro Hyp Gly Cpg Ser Dtic Cpg; and ii)Acetyl Lys Lys Arg Pro Hyp Gly Cpg Ser Dtic Cpg.
 11. A method of makinga sustained release complex comprising the steps of mixing a protein orpeptide with a gallic acid ester, and isolating the precipitate.
 12. Themethod of claim 11, wherein the complex is formed between pH from pH 6.5to 8.6.
 13. The method of claim 11 where the gallic acid ester is PGG.14. The method of claim 11 wherein the gallic acid ester is EGCG.
 15. Amethod of administering a sustained release complex wherein the complexcomprises a pharmaceutically acceptable formulation of a complex of aprotein or peptide and a gallic acid ester.
 16. The method of claim 15where the gallic acid ester is PGG.
 17. The method of claim 15 whereinthe gallic acid ester is EGCG.
 18. The method of claim 15 wherein saidpeptide is a B1 peptide antagonist.
 19. The method of claim 16 whereinsaid peptide is selected from i) DOrn Lys Arg Pro Hyp Gly Cpg Ser DticCpg; and ii) Acetyl Lys Lys Arg Pro Hyp Gly Cpg Ser Dtic Cpg whereinDOrn is the D isomer of ornithine, Hyp is Trans-4-hydroxy-proline, Dticis the D isomer of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, andCpg is cyclopentylglycine.
 20. The method of claim 19 wherein saidpeptide is selected from i) DOrn Lys Arg Pro Hyp Gly Cpg Ser Dtic Cpg;and ii) Acetyl Lys Lys Arg Pro Hyp Gly Cpg Ser Dtic Cpg.